Synergistic Multi-Enzyme Cascades Assembled on Modular Protein Scaffolds for Efficient PET Biorecycling

Abstract

The global crisis of polyethylene terephthalate (PET) waste demands new strategies for sustainable management. Biocatalytic recycling offers a promising route, but direct depolymerization under moderate conditions is often limited by kinetic constrains of individual enzyme. Here, we engineer and optimize modular protein scaffolds that utilize orthogonal interaction domains, including the cohesin-dockerin pairs from bacterial cellulosomes and the SH3 ligand/domain pair from mouse Crk, to assemble complementary hydrolases (PETase, MHETase, and ICCG) into a synergistic multi-enzyme cascade. This architecture overcomes intermediate inhibition, efficiently converting PET hydrolysis products into the final monomers, terephthalic acid (TPA) and ethylene glycol (EG), outperforming previous multi-enzyme systems. The systems application is further evaluated by stability enhancement via immobilization on metal-organic frameworks (MOFs), coupling depolymerization to the valorization of EG into glycolic acid (GA), and adapting the system for scalable whole-cell biocatalysis. This scaffold-based multi-enzyme approach provides a new strategy for developing integrated biocatalytic systems to advance a circular PET economy.